New vectors for co-expression of proteins: Structure of Bacillus subtilis ScoAB obtained by high-throughput protocols

2007 ◽  
Vol 53 (2) ◽  
pp. 396-403 ◽  
Author(s):  
Lucy Stols ◽  
Min Zhou ◽  
William H. Eschenfeldt ◽  
Cynthia Sanville Millard ◽  
James Abdullah ◽  
...  
PLoS ONE ◽  
2014 ◽  
Vol 9 (2) ◽  
pp. e88207 ◽  
Author(s):  
Lingyan Zhu ◽  
Qing Xu ◽  
Ling Jiang ◽  
He Huang ◽  
Shuang Li

2007 ◽  
Vol 73 (20) ◽  
pp. 6436-6443 ◽  
Author(s):  
Andreas Urban ◽  
Stefan Eckermann ◽  
Beate Fast ◽  
Susanne Metzger ◽  
Matthias Gehling ◽  
...  

ABSTRACT Cells containing reporters which are specifically induced via selected promoters are used in pharmaceutical drug discovery and in environmental biology. They are used in screening for novel drug candidates and in the detection of bioactive compounds in environmental samples. In this study, we generated and validated a set of five Bacillus subtilis promoters fused to the firefly luciferase reporter gene suitable for cell-based screening, enabling the as yet most-comprehensive high-throughput diagnosis of antibiotic interference in the major biosynthetic pathways of bacteria: the biosynthesis of DNA by the yorB promoter, of RNA by the yvgS promoter, of proteins by the yheI promoter, of the cell wall by the ypuA promoter, and of fatty acids by the fabHB promoter. The reporter cells mainly represent novel antibiotic biosensors compatible with high-throughput screening. We validated the strains by developing screens with a set of 14,000 pure natural products, representing a source of highly diverse chemical entities, many of them with antibiotic activity (6% with anti-Bacillus subtilis activity of ≤25 μg/ml]). Our screening approach is exemplified by the discovery of classical and novel DNA synthesis and translation inhibitors. For instance, we show that the mechanistically underexplored antibiotic ferrimycin A1 selectively inhibits protein biosynthesis.


Author(s):  
P. Opdensteinen ◽  
S. J. Dietz ◽  
B. B. Gengenbach ◽  
J. F. Buyel

Biofilm-forming bacteria are sources of infections because they are often resistant to antibiotics and chemical removal. Recombinant biofilm-degrading enzymes have the potential to remove biofilms gently, but they can be toxic toward microbial hosts and are therefore difficult to produce in bacteria. Here, we investigated Nicotiana species for the production of such enzymes using the dispersin B-like enzyme Lysobacter gummosus glyco 2 (Lg2) as a model. We first optimized transient Lg2 expression in plant cell packs using different subcellular targeting methods. We found that expression levels were transferable to differentiated plants, facilitating the scale-up of production. Our process yielded 20 mg kg−1 Lg2 in extracts but 0.3 mg kg−1 after purification, limited by losses during depth filtration. Next, we established an experimental biofilm assay to screen enzymes for degrading activity using different Bacillus subtilis strains. We then tested complex and chemically defined growth media for reproducible biofilm formation before converting the assay to an automated high-throughput screening format. Finally, we quantified the biofilm-degrading activity of Lg2 in comparison with commercial enzymes against our experimental biofilms, indicating that crude extracts can be screened directly. This ability will allow us to combine high-throughput expression in plant cell packs with automated activity screening.


Microbiology ◽  
2010 ◽  
Vol 156 (6) ◽  
pp. 1600-1608 ◽  
Author(s):  
Eric Botella ◽  
Mark Fogg ◽  
Matthieu Jules ◽  
Sjouke Piersma ◽  
Geoff Doherty ◽  
...  

Plasmid pBaSysBioII was constructed for high-throughput analysis of gene expression in Bacillus subtilis. It is an integrative plasmid with a ligation-independent cloning (LIC) site, allowing the generation of transcriptional gfpmut3 fusions with desired promoters. Integration is by a Campbell-type event and is non-mutagenic, placing the fusion at the homologous chromosomal locus. Using phoA, murAA, gapB, ptsG and cggR promoters that are responsive to phosphate availability, growth rate and carbon source, we show that detailed profiles of promoter activity can be established, with responses to changing conditions being measurable within 1 min of the stimulus. This makes pBaSysBioII a highly versatile tool for real-time gene expression analysis in growing cells of B. subtilis.


PLoS Biology ◽  
2016 ◽  
Vol 14 (1) ◽  
pp. e1002341 ◽  
Author(s):  
Alexander J. Meeske ◽  
Christopher D. A. Rodrigues ◽  
Jacqueline Brady ◽  
Hoong Chuin Lim ◽  
Thomas G. Bernhardt ◽  
...  

Author(s):  
Marie-Laure Diebold-Durand ◽  
Frank Bürmann ◽  
Stephan Gruber

2019 ◽  
Author(s):  
Adrian Sven Geissler ◽  
Christian Anthon ◽  
Enrique González-Tortuero ◽  
Line Dahl Poulsen ◽  
Thomas Beuchert Kallehauge ◽  
...  

AbstractThe genome of Bacillus subtilis continues to provide exiting genomic insights. However, the growing collective genomic knowledge about this micro-organism is spread across multiple annotation resources. Thus, the full annotation is not directly accessible neither for specific genes nor for large-scale high-throughput analyses. Furthermore, access to annotation of non-coding RNA genes (ncRNAs) and polycistronic mRNAs is difficult. To address these challenges we introduce the Bacillus subtilis genome atlas, BSGatlas, in which we integrate and unify multiple existing annotation resources. Our integration provides twice as many ncRNAs than the individual resources, improves the positional annotation for 70% of the combined ncRNAs, and makes it possible to infer specific ncRNA types. Moreover, we unify known transcription start sites, termination, and transcriptional units (TUs) as a comprehensive transcript map. This transcript map implies 815 new TUs and 6, 164 untranslated regions (UTRs), which is a five-fold increase over existing resources. We furthermore, find 2, 309 operons covering the transcriptional annotation for 93% of all genes, corresponding to an improvement by 11%. The BSGatlas is available in multiple formats. A user can either download the entire annotation in the standardized GFF3 format, which is compatible with most bioinformatics tools for omics and high-throughput studies, or view the annotation in an online browser at http://rth.dk/resources/bsgatlas.ImportanceThe Bacillus subtilis genome has been studied in numerous context and consequently multiple efforts have been made in providing a complete annotation. Unfortunately, a number of resources are no longer maintained, and (i) the collective annotation knowledge is dispersed over multiple resources, of which each has a different focus of what type of annotation information they provide. (ii) Thus, it is difficult to easily and at a large scale obtain information for a genomic region or genes of interest. (iii) Furthermore, all resources are essentially incomplete when it comes to annotating non-coding and structured RNA, and transcripts in general. Here, we address all three problems by first collecting existing annotations of genes and transcripts start and termination sites; afterwards resolving discrepancies in annotations and combining them, which doubled the number of ncRNAs; inferring full transcripts and 2,309 operons from the combined knowledge of known transcript boundaries and meta-information; and critically providing it all in a standardized UCSC browser. That interface and its powerful set of functionalities allow users to access all the information in a single resource as well as enables them to include own data on top the full annotation.


Sign in / Sign up

Export Citation Format

Share Document